The bacteria of the Bacillus genus are used extensively in the production of different industrially significant enzymes. The most significant production hosts are B. amyloliquefaciens and B. licheniformis that are used as producers of proteases and amylases, for instance. Industrial processes generate significant amounts of bacterial mass that needs to be inactivated prior to its discharge into the environment, especially if the bacterium is a genetically engineered bacterium. Destroying sporulated production cells requires more intensive processing than asporogenous cells. The spores of Bacillus endure heat much better than vegetative cells and therefore, destroying them by heating requires high temperatures and long-term treatments. These treatments invariably increase the equipment and operating costs in the production. This is why it is desirable to use a non-sporulating Bacillus strain. The present invention provides a solution to the sporulation problem that significantly improves the use of B. subtilis as a production organism.
Sporulation is a multistage (I to VII) event that is initiated in certain growth conditions in which first a pre-spore is created inside the mother cell. Finally, the mother cell dies and the mature spore is freed. The spore can endure a higher degree of dehydration and heat than the mother cell and thus ensures the survival of the bacterium in unfavourable conditions. In favorable conditions, the spore is activated and the division of the bacterial cell is restarted. The different stages of sporulation have been established by research on gene mutations affecting sporulation. Over 125 genes affecting sporulation are known (Stragier and Losick, 1996).
The production of proteins using a Bacillus bacterium incapable of sporulation is disclosed in WO97/03185, for instance, which proposes the elimination of sporulation by mutating the sporulation genes. The publication describes the deletion of the sporulation gene spoIIAC from Bacillus licheniformis. The deletion was performed using a temperature-sensitive plasmid to which a PCR product prepared by the SOE (splicing by overlap extension) technique was introduced, in which product the regions on both sides of the spoIIAC gene were joined together in such a manner that the spoIIAC gene in the middle was removed. The SOE technique is described in U.S. Pat. No. 5,023,171, for instance. The in vitro deletion mutation can be introduced inside the bacterial cell by means of the plasmid, and the replication of the free plasmid can be prevented by raising the temperature, thus revealing the recombinant bacteria in which the plasmid is inserted in the chromosome. The deletion of the desired gene takes place when the plasmid detaches from the chromosome in a certain manner. In said WO patent publication, the described technique was, however, unable to delete the spoIIAC gene from the B. subtilis bacterium. The recombination at the detaching stage of the plasmid always occurred in such a manner that the spoIIAC gene remained intact.
Non-sporulating B. subtilis strains are, however, described elsewhere. EP 164,117 relates to a B. subtilis strain with a mutation in the spoIIA gene, EP 492,274 relates to a B. subtilis strain with a mutation in the spoIID gene, and U.S. Pat. No. 4,450,235 and U.S. Pat. No. 4,450,236 relate to a B. subtilis strain with a deletion in the spoOA gene. Said genes are associated with sporulating stage II or an earlier stage.
One of the known genes of the next sporulating stage (III) is the sigG gene (=spoIIIG gene) that codes the sigma-G factor which binds to an RNA polymerase that in turn can bind to the promoter of certain sporulating genes in the pre-spore. This sigma-G factor is necessary at the third stage of sporulation, and it is known to control at least 19 gene transcriptions (Ishii et al., 2001). The products of the genes associated with the Sigma-G control system improve the survival ability and re-germination of the spores (Haldenwang, 1995).
Fougler and Errington (1989) have described a B. subtilis 646 strain that is a spontaneously formed sigG mutant. The only thing known of the mutation is that it is outside the promoter and 30 first codons. This information has been obtained from an active spoIIIG-lacZ fusion that was inserted into the chromosome. In the case of such spontaneous or induced random mutants, the location or exact action of the mutation is usually not known. The reverse-mutation possibility, i.e. the reversal of sporulation as it was, of such strains cannot be controlled and the possibility of various suppressor mutations is also high. Since the location of the mutation is not known, it is usually not possible, either, to know whether a changed protein is created. In addition, a mutation in another gene can, for instance, suppress the effect of the original mutation. Attempts have also been made to inactivate the sigG gene by making insertions, for instance. Illing et al. (1990), for instance, cloned a HindIII-Pst1 fragment of 320 base pairs from sigG to an integration plasmid and obtained as a result of the integration a non-sporulating B. subtilis strain N15 (trpC2 spoIIIG::pSGMU422). The integration plasmid, however, detached from the chromosome without selection pressure with chloramphenicol, and consequently, this type of strain is not suitable as a production host.
Karmazyn-Campelli et al. (1989) have described the inactivation of the sigG gene by inserting a 1.5-kb chloramphenicol resistance cassette (cat) between codons 166 and 167 (spoIIIG::cat). This insertion was, however, done in such a manner that the sigG gene still remained, even though cut, in the chromosome. Thus, there is a risk of reverse-mutation. Even though a strain inactivated by cat insertion cannot recombine, the deletion may return the activity of the sigG gene and the sporulation of the strain back to normal.
Karmazyn-Campelli et al. (1989) have further deleted a fragment of the sigG gene, 70 base pairs, between codons 18 and 42. The effect of this deletion (spoIIIGΔ1) on sporulation remains, however, unclear, because during the deletion, an extra HaeIII fragment of 18 base pairs unexpectedly appeared between said codons from pUC8 used as the cloning vector. This extra fragment had a stop codon right at the beginning, so there is no certainty that the deletion of 70 base pairs is sufficient for ending sporulation. Without the extra fragment, at least a partly functioning sigma factor could be created even after the deletion, especially since the deletion does not include regions which are believed to be significant in binding to target promoters. In other words, it did not become clear, whether the inactivation of the sigG gene was caused by the deletion or merely by the extra stop codon.
Kim J.-H. et al. 2001 have described a B. subtilis mutant with a deletion in the spoIIIG gene. The deletion in question was only one nucleotide long and resided outside the functional regions of the gene (sigG gene nucleotide 397, tymidine (T) deleted). A kanamycin-resistance block (kanar) was inserted after the deletion site. This produced mutants with a reduced sporulation capability. There is no information on the stability of the strain. The reversion frequency of one nucleotide is, however, significant, and this kind of strain is, therefore, not recommended as the production host of a foreign protein.
Studies published earlier have usually examined the different stages of sporulation and related mutants, without focusing on their stability. Consequently, none of the above sigG mutants of the B. subtilis bacterium are suitable as a production strain, of which a permanent, i.e. non-reversible, sporulation mutation is required. The present invention now provides a fully non-sporulating, stable B. subtilis strain suitable as a production strain. In addition, the sporulation gene is deleted in such a manner that it has no unfavourable effects on the production of the desired product or its properties.